This application addresses the broad Challenge Area (08), "Genomics", and the specific Challenge Topic (08-ES-106), "The role of environmental exposure in copy number variation (CNV)". CNV events are deletions or duplications that are too small to be recognized by karyotypic analysis. Spontaneous CNVs have been recently recognized as a major source for sporadic disease in human populations, perhaps most notably in Autism Spectrum Disorders (ASD). The mechanisms underlying the formation of CNVs are not known, although it is likely that both environmental and intrinsic cellular factors affect the rate of CNVs. In this proposal, we describe the use of the yeast Saccharomyces cerevisiae as a model to find out how environmental contaminants and various mutations control the rate of CNVs. We will examine both large (>1kb deletions or duplications) and small (1- 1000 bp) CNV events. To investigate the effect of environmental factors and contaminants on large CNV events (Specific Aim 1), we will use a reporter in which duplications can be selected and deletions can be detected by screening. We will use a cassette that contains two yeast genes SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. Using yeast strains in which this cassette is integrated into various positions, we will measure the rate of cassette amplifications in a wild-type strain growing under standard lab conditions. We will then determine the rate of amplifications in response to various DNA damaging agents, and common agrochemicals. We will also optimize a high-throughput version of the formaldehyde-copper resistance assay for use in preliminary screens of diverse chemical libraries to identify unknown environmental contaminants associated with CNV stimulation. To determine which mechanisms are involved in the amplifications (homologous recombination between dispersed repeats, microhomology- mediated recombination, non-homologous end joining or aneuploidy), we will characterize the strains with CNV events using DNA microarrays and a gel system that separates intact chromosomal DNAs (CHEF gels). In Specific Aim 2, we will conduct an investigation of cellular mechanisms linked to CNV formation, focusing on spontaneous events and those stimulated by low-dose ionizing radiation. We will determine the rates of CNV and the types of alterations in cells exposed to radiation at different stages of the cell cycle. In addition, we will examine spontaneous CNVs in meiotic cells. A set of mutant strains with compromised DNA repair and replication will also be examined to characterize the genetic control of CNV formation. In Specific Aim 3, we will investigate a different class of CNV events, those that duplicate or delete less than 1000 bp. For this analysis, we will use high-throughput DNA sequencing of sub-cultured untreated yeast strains and of strains treated with low levels of radiation. The analysis should provide much needed insight into the prevalence of this class of CNV, as well as into the mechanism of its formation. In this proposal, we examine the effects of environmental stresses and alterations of the genetic background on the frequency of de novo copy number variation (deletions and duplications of DNA sequences) in yeast. During the past few years, it has become increasingly clear that copy number variation (CNV) is associated with many human diseases. Thus, factors that elevate the rate of CNV are likely to elevate the frequency of those diseases.